CN104694526B - Catalytic esterification and the Sn-1,3 selectivity immobilized lipase of transesterification and preparation method thereof - Google Patents
Catalytic esterification and the Sn-1,3 selectivity immobilized lipase of transesterification and preparation method thereof Download PDFInfo
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- CN104694526B CN104694526B CN201310656064.2A CN201310656064A CN104694526B CN 104694526 B CN104694526 B CN 104694526B CN 201310656064 A CN201310656064 A CN 201310656064A CN 104694526 B CN104694526 B CN 104694526B
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- enzyme
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- immobilized
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- HPGGPRDJHPYFRM-UHFFFAOYSA-J tin(iv) chloride Chemical compound Cl[Sn](Cl)(Cl)Cl HPGGPRDJHPYFRM-UHFFFAOYSA-J 0.000 description 2
- 229960001124 trientine Drugs 0.000 description 2
- 238000000108 ultra-filtration Methods 0.000 description 2
- 238000001291 vacuum drying Methods 0.000 description 2
- 239000011592 zinc chloride Substances 0.000 description 2
- 235000005074 zinc chloride Nutrition 0.000 description 2
- WVICRWJHVDRYSU-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;methyl prop-2-enoate Chemical compound COC(=O)C=C.C=CC1=CC=CC=C1C=C WVICRWJHVDRYSU-UHFFFAOYSA-N 0.000 description 1
- GJOHLWZHWQUKAU-UHFFFAOYSA-N 5-azaniumylpentan-2-yl-(6-methoxyquinolin-8-yl)azanium;dihydrogen phosphate Chemical group OP(O)(O)=O.OP(O)(O)=O.N1=CC=CC2=CC(OC)=CC(NC(C)CCCN)=C21 GJOHLWZHWQUKAU-UHFFFAOYSA-N 0.000 description 1
- 102000057234 Acyl transferases Human genes 0.000 description 1
- 108700016155 Acyl transferases Proteins 0.000 description 1
- 108010006591 Apoenzymes Proteins 0.000 description 1
- 238000009010 Bradford assay Methods 0.000 description 1
- 241000222173 Candida parapsilosis Species 0.000 description 1
- 239000004215 Carbon black (E152) Substances 0.000 description 1
- 241000222175 Diutina rugosa Species 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 244000068988 Glycine max Species 0.000 description 1
- 235000010469 Glycine max Nutrition 0.000 description 1
- 108090000604 Hydrolases Proteins 0.000 description 1
- 102000004157 Hydrolases Human genes 0.000 description 1
- 241000235395 Mucor Species 0.000 description 1
- 238000005481 NMR spectroscopy Methods 0.000 description 1
- 102000019280 Pancreatic lipases Human genes 0.000 description 1
- 108050006759 Pancreatic lipases Proteins 0.000 description 1
- 229920002319 Poly(methyl acrylate) Polymers 0.000 description 1
- 239000004743 Polypropylene Substances 0.000 description 1
- QOSMNYMQXIVWKY-UHFFFAOYSA-N Propyl levulinate Chemical compound CCCOC(=O)CCC(C)=O QOSMNYMQXIVWKY-UHFFFAOYSA-N 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical class [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 241000223258 Thermomyces lanuginosus Species 0.000 description 1
- 230000004308 accommodation Effects 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 150000001252 acrylic acid derivatives Chemical class 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 125000001931 aliphatic group Chemical group 0.000 description 1
- 239000003957 anion exchange resin Substances 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 239000011230 binding agent Substances 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 229940055022 candida parapsilosis Drugs 0.000 description 1
- 238000007385 chemical modification Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000010924 continuous production Methods 0.000 description 1
- 238000007334 copolymerization reaction Methods 0.000 description 1
- 238000005034 decoration Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 239000002270 dispersing agent Substances 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- 238000004821 distillation Methods 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000003778 fat substitute Substances 0.000 description 1
- 235000013341 fat substitute Nutrition 0.000 description 1
- 235000019387 fatty acid methyl ester Nutrition 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 238000005194 fractionation Methods 0.000 description 1
- 238000007306 functionalization reaction Methods 0.000 description 1
- 230000013595 glycosylation Effects 0.000 description 1
- 238000006206 glycosylation reaction Methods 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 235000020256 human milk Nutrition 0.000 description 1
- 210000004251 human milk Anatomy 0.000 description 1
- 229930195733 hydrocarbon Natural products 0.000 description 1
- 238000005984 hydrogenation reaction Methods 0.000 description 1
- 230000005660 hydrophilic surface Effects 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 238000009533 lab test Methods 0.000 description 1
- 230000002045 lasting effect Effects 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- JRZJOMJEPLMPRA-UHFFFAOYSA-N olefin Natural products CCCCCCCC=C JRZJOMJEPLMPRA-UHFFFAOYSA-N 0.000 description 1
- 235000020660 omega-3 fatty acid Nutrition 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 229940097411 palm acid Drugs 0.000 description 1
- 210000000496 pancreas Anatomy 0.000 description 1
- 229940116369 pancreatic lipase Drugs 0.000 description 1
- 230000000704 physical effect Effects 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 229920003023 plastic Polymers 0.000 description 1
- 229920000333 poly(propyleneimine) Polymers 0.000 description 1
- 229920001155 polypropylene Polymers 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 238000007670 refining Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 235000021391 short chain fatty acids Nutrition 0.000 description 1
- 150000004666 short chain fatty acids Chemical class 0.000 description 1
- 238000007086 side reaction Methods 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 235000002316 solid fats Nutrition 0.000 description 1
- 229940126680 traditional chinese medicines Drugs 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 238000009777 vacuum freeze-drying Methods 0.000 description 1
- 239000003981 vehicle Substances 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
- C12P7/6409—Fatty acids
- C12P7/6418—Fatty acids by hydrolysis of fatty acid esters
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
- C12N11/082—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer obtained by reactions only involving carbon-to-carbon unsaturated bonds
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
- C12N11/082—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer obtained by reactions only involving carbon-to-carbon unsaturated bonds
- C12N11/087—Acrylic polymers
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
- C12P7/6436—Fatty acid esters
- C12P7/6445—Glycerides
- C12P7/6458—Glycerides by transesterification, e.g. interesterification, ester interchange, alcoholysis or acidolysis
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/10—Biofuels, e.g. bio-diesel
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Oil, Petroleum & Natural Gas (AREA)
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The present invention provides a kind of catalytic esterifications and the Sn-1,3 selectivity immobilized lipase of transesterification and preparation method thereof.Specifically, the present invention relates to a kind of immobilized lipase, it is it includes (a) functional group and C8‑15The connection of alkane end primary amine groups carrier, the content of the functional group is 0.5~6mmol/g, and pore-size distribution is 12~80nm；(b) it is fixed on the lipase of the carrier.The present invention also provides the applications and preparation of the preparation method of the immobilized lipase, its application and application method, kit or product and the carrier.Immobilized lipase of the invention has both high ester exchange activity, strong Sn-1,3 specificities and high esterification ability, is with a wide range of applications.
Description
Technical field
The invention belongs to food and enzyme engineering field.More particularly it relates to for have both esterification, transesterification is urged
Change the Sn-1,3 selected fat enzyme immobilizatio method and its application of function.
Background technique
Lipase (lipase, EC3.1.1.3) full name is glycerol trigalloyl ester hydrolase, it can be specifically in grease circle
Catalytic activity is played on face, industrially has huge application in food, papermaking, leather, detergent, pharmacy etc..Different strain
The lipase in source can also have catalytic esterification, transesterification, alcoholysis, mesotomy etc. other than catalyzing oil hydrolysis
Function.However, natural fat enzyme stability is poor, is difficult to purify after reaction, cannot reuse, this not only lowers the uses of enzyme
Efficiency improves production cost, and is difficult to realize continuous operation, limits its application industrially.Pass through immobilization
Enzyme figuration on the carrier with certain mechanical strength, is become a kind of effective way for solving above-mentioned production bottleneck by technology.
Enzyme immobilization technology is to be fettered or be limited to certain area by physics or chemical means for the enzyme to dissociate
Interior, making it still can be carried out distinctive catalysis reaction and can recycle a kind of technology reused.Due to above-mentioned advantage, so that very
More researchers expand the careful research to immobilised enzymes.In 1967, immobilised enzymes was used in fractionation amino for the first time
In sour industry.Recently as the development of organic chemistry, protein chemistry, material science etc., enzyme immobilization technology achieves length
The progress of foot.
It is strong to be respectively as follows: high ester exchange capacity Sn-1,3 specificity for most widely used immobilized lipase in grease field
Immobilized lipase；And it is used to prepare the immobilized lipase of the high esterification activity of aliphatic ester.Generally select immobilization tool
Standby high ester exchange activity and Sn-1,3 specific lipase can be selected from (but being not limited to): dredge the thermophilic hyphomycete of cotton like
(Thermomyces lanuginose), rhizomucor miehei (Rhizomucor miehei), rice black wool mould (Mucor
Miehei), pseudomonad (Pseudomonas sp.)/head mold (Rhizopus sp.), aspergillus niger (Aspergillus
) and Burkholderia (Burkholderia sp.) niguer.And for the application in terms of short chain fatty acids Lipase absobed, then it selects
It selects such as (but not limited to): antarctic candida B (Candida Antarctica B)/antarctic candida A (Candida
Antarctica A), fold candida (Candida rugosa) etc..
CarlaEt al. (" Production of human milk fat substitutes enriched
in omega-3 polyunsaturated fatty acids using immobilized commercial lipases
And Candida parapsilosis lipase/acyltransferase ", Journal of Molecular
Catalysis B:Enzymatic, 65 (2010) 122-127) it has studied to be rich in sweet three ester of palmitinic acid and oleic acid as reaction
Substrate, with Lipozyme TL IM (by Novi letter (Novozyme) joint-stock company produce a kind of immobilized lipase),
(these three enzymes are the immobilization rouge that letter (Novozyme) joint-stock company of Novi produces by Lipozyme RM IM, Novozyme435
Fat enzyme) it is catalyst preparation OPO (bis- oleic acid -2- palmitic acid of 1,3-, three ester).Research is found: in terms of 2 insertion rates, instead
Novozyme435 shows poor specificity when answering first, and (33.2% mole of more oleic acid is inserted on the position Sn-2
Than), it was demonstrated that it does not have specificity when acidolysis.Also, in research it has also been found that after reaction a batch, in addition to Lipozyme
RM IM, which reacts 10 batches, still to be kept except preferable vigor, and in addition the remaining enzyme activity of two kinds of enzymes is under the increase all of reaction times
Drop quickly, Lipozyme TL IM remaining vigor after reaction 10 batches is only 20% or so.
Igor Nascentes dos Santos Corr ê a et al. (" Enzymatic Biodiesel Synthesis
Using a Byproduct Obtained from Palm Oil Refining ", Enzyme Research, 2011, article
ID:814507 it) has studied with palm oil fatty acid deodorization distillate (PFAD, palm oil fatty acid distillate)
It is reaction substrate with short chain alcohol, prepares single alkane of fatty acid as catalyst using Lipozyme TL IM, Lipozyme RM IM
Base ester.Result of study discovery: when using methanol as acry radical donor, the conversion ratio that Lipozyme RM IM catalysis generates methyl esters is
The conversion ratio that 52.3%, Lipozyme TL IM catalysis generate methyl esters is 28.8%, it was demonstrated that Lipozyme TL IM and
Lipozyme RM IM is not suitable for catalytic esterification.
(" the Different enzyme requirements for the such as Hern á ndez-Mart í n E
Synthesisi of biodiesel Novozyme435and Lipozyme TL IM ", Bioresource
Technology, 99 (2008) 277-286) it discloses and adds quantitative response for 24 hours by the enzyme of substrate, 10%W/W of sweet three ester,
10% enzyme activity is only remained after Lipozyme TL IM reaction is primary for the second time.This illustrates that Lipozyme TL IM is being reused
Property on exist very big limitation.
(the Improvement in lipase-catalyzed synthesis of such as Mohamed M.Soumanou
Fatty acid methyl esters from sunflower oil, enzyme and Microbial Technology,
33 (2003) 97-103) it discloses using sunflower oil as substrate, methanol is added batch-wise, for 24 hours, Lipozyme TL IM passes through for reaction
Vigor is only left less than 30% after 8 batch reactions.The result of study further demonstrates the sight of Hern á ndez-Mart í n E et al.
Point.
The method of many immobilized lipases is all to be incited somebody to action using macromolecule resin as carrier by certain technological means at present
Lipase is carried on resin.For example, describing one kind in the patent that patent announcement number is EP0140542B1 about rice is black
Root Mucor yielding lipase is fixed on macroporous weakly basic anion exchange resin, and is used for transesterification or acidolysis reaction.
Xie Zhidong etc. (" immobilization research of the polymethyl acrylate class macroreticular resin to porcine pancreatic lipase ", ion exchange and absorption,
1995,11 (1), 24~29) the macroporous acrylic methyl esters-divinylbenzene crosslink copolymer synthesized using oneself is carrier to pig pancreas rouge
Fat enzyme has carried out immobilization, and hydrolyzes vigor to it and investigated.
Although there is certain research to fatty enzyme immobilizatio in the prior art, the ultimate challenge faced now is: arriving
So far there are no a kind of immobilised enzymes can simultaneously catalytic transesterification and esterification, and also have stronger specificity,
Which limits immobilised enzymes in fats and oils processing industry, such as the application in biodiesel, functional grease, fine chemical product.
Therefore, in this field there is an urgent need to develop go out can simultaneously catalytic transesterification and esterification and also have it is stronger
Immobilised enzymes of specificity and preparation method thereof.
Summary of the invention
The main object of the present invention be that offer can simultaneously catalytic transesterification and esterification and also have it is stronger specially
The immobilised enzymes and preparation method thereof of one property (such as Sn-1 selectivity).Another object of the present invention is to provide fixations of the invention
Change application of the enzyme in catalytic transesterification, esterification, acidolysis and/or alcoholysis reaction, and uses immobilized enzyme catalysis of the invention
The method of these reactions.Kit or product comprising immobilized lipase of the present invention are additionally provided in the present invention.In addition, this hair
Bright another object also resides in the application for providing deacidite carrier in preparation immobilized lipase of the present invention.
In the first aspect of the invention, a kind of immobilized lipase is provided, it includes:
(a) carrier, wherein the functional group of the carrier is and C8-15Alkane end connection primary amine groups, the function
Group content is 0.5~6mmol/g, and pore-size distribution is 12~80nm；
(b) it is fixed on the lipase of the carrier.
In some instances, the functional group content of the carrier is 1~5mmol/g, and pore-size distribution is 15~60nm, excellent
Choosing, the particle diameter distribution of the carrier is 0.1~0.4mm, and preferably 0.15~0.3mm, specific surface area is 100~600m2/ g,
Preferably 150~500m2/g。
In some instances, the carrier is deacidite carrier, and preferably weak-base ion-exchange resin carries
Body.
In some examples of the invention, the skeleton of the carrier is polyacrylate or styrene type skeleton.
In other examples, the carrier is alkalescent acrylate ionomer exchanger resin or alkalescent polystyrene ion
Exchanger resin.
In other examples, the carrier is made of material selected from the group below: the cross-linked polymer of acrylate；Crosslinking
Polymethyl methacrylate；Methacrylate, dimethyl maleate, allyl acetate, acrylonitrile or methyl ethylene
The copolymer of ketone and styrene or divinylbenzene；Acrylate or methacrylate and ethyleneglycol dimethyacrylate
Cross-linked copolymer；Or the copolymer of styrene and divinylbenzene.
In other examples, the carrier is methyl acrylate-divinylbenzene copolymer or styrene and divinylbenzene
Copolymer.
In some instances, the deacidite is alkalescent acrylate ionomer exchanger resin, it is preferred that institute
State alkalescent acrylate ionomer exchanger resin to prepare using following methods: (i) preparation acrylic compounds-divinylbenzene is total to
Polymers；(ii) it is swollen the copolymer, and amination is carried out to it.
In other examples, the deacidite is by methyl acrylate (or hydroxy-ethyl acrylate, propylene
Sour polyglycidyl, methyl methacrylate, hydroxyethyl methacrylate, glycidyl methacrylate) and divinylbenzene with 20:
The mass ratio (preferably 15:1~10:1) of 1~1:1, pore-foaming agent (such as the mixture or gasoline of toluene and liquid wax, normal heptane,
Butyl acetate, 1,2- dichloroethanes) and initiator () benzoyl peroxide, azo-bis-isobutyl cyanide or potassium peroxydisulfate) existing
In the case of polymerize, the ratio of copolymer and pore-foaming agent therein is 3:1~1:3 (preferably 3:2~3:4), and initiator is above-mentioned body
0.1% (mass ratio) of system.The polymerization temperatures range of above-mentioned monomer is 60~100 DEG C, preferably 65~85 DEG C.Gained copolymer with
Sweller (or mixtures thereof N,N-dimethylformamide, nitrobenzene, methylene chloride, dichloroethanes, carbon disulfide, dioxane)
It is swollen with the volume ratio (preferably 1:2~1:10) of 1:1~1:15, swelling condition can are as follows: at room temperature, copolymer is in sweller
Middle immersion is swollen 4~24 hours, preferably 6~16 hours.By copolymer and 1,8- octamethylenediamine with methyl esters key and 1,8- in copolymer
The molar ratio of octamethylenediamine is with the ratio of 1:1~1:10 (preferably 1:2~1:5), under 100~150 DEG C (preferably 120~140 DEG C)
Amination, aminating reaction time are 4~24 hours (preferably 8~12 hours).
In some instances, the deacidite is alkalescent polystyrene ion-exchange resin, it is preferred that
The alkalescent polystyrene ion-exchange resin is prepared using following methods: (i') prepares polystyrene microsphere；(ii') it is swollen
The microballoon, and it is acylated that chlorine is carried out to it；(iii') it is swollen the chlorine acylated resin microballoon, and amination is carried out to it.
In some instances, the deacidite is by styrene, divinylbenzene and pore-foaming agent (e.g., toluene
With mixture or gasoline, normal heptane, butyl acetate, 1, the 2- dichloroethanes of liquid wax, preferably toluene and liquid wax 2:1 volume ratio
Mixture) in the presence of initiator (such as benzoyl peroxide, azo-bis-isobutyl cyanide, potassium peroxydisulfate, preferably benzoyl peroxide)
Under prepare polystyrene microsphere.Wherein, the mass ratio of styrene and divinylbenzene is 5:1~1:2, preferably 3:1~1:1；Benzene second
The mass ratio of alkene and divinylbenzene and pore-foaming agent is 1:1~1:6, preferably 1:1.5~1:4.The polymerization temperatures range of above-mentioned monomer
It is 60~95 DEG C, preferably 80~90 DEG C.Polymerization time range is 4~10 hours, preferably 6~8 hours.Gained polystyrene it is micro-
Ball (is preferably but not limited to methylene chloride, N, N- dimethyl formyl in 1:1~1:15 volume ratio, the preferably sweller of 1:2~1:10
Or mixtures thereof amine, nitrobenzene, methylene chloride, dichloroethanes, carbon disulfide, dioxane) in swelling.Swelling condition are as follows: room
Under temperature, copolymer impregnates in a solvent, is swollen 4~24 hours, preferably 6~16 hours.Benzal and 9- chlorine in above-mentioned microballoon
The molar ratio of pelargonyl chloride is 1:0.1~1:2, preferably 1:0.2~1:1, and 9- chlorine pelargonyl chloride and catalyst are (anhydrous zinc chloride, anhydrous
Stannic chloride, preferably anhydrous aluminum chloride) molar ratio be 1:0.1~1:2, preferably 1:0.2~1:1,30~75 DEG C (preferably 35~
50 DEG C) under carry out chlorine acylated, 2~12h of reaction time, preferably 3~8h.By resulting chlorine acylated resin microballoon in 1:1~1:15
Sweller (N,N-dimethylformamide, nitrobenzene, methylene chloride, dichloroethanes, two sulphur of volume ratio (preferably 1:2~1:10)
Change carbon, or mixtures thereof dioxane) in the presence of be swollen 4~10 hours, preferably 6~8 hours.With ammonium hydroxide, ethylenediamine, divinyl
Triamine, triethylene tetramine or the tetraethylenepentamine (mole and NH of chlorine in chlorine acylated resin3Mole ratio is excellent in 1:1~1:20
Select 1:8~1:15) at 40~80 DEG C, preferably 50~60 DEG C progress aminations, the aminating reaction time 4~24 hours, preferably 10~14
Hour.
In certain embodiments of the present invention, the functional group content of the carrier is 1~5mmol/g, pore-size distribution
For 15~60nm, particle diameter distribution is 0.15~0.3mm, and specific surface area is 150~500m2/g。
In some instances, the carrier is WR-8-1, WR-8-2, WR-8-3, WR-8-4, WR-8-5, WR-8-6, WR-
8-7、WR-8-8、WR-8-9、WR-8-10、WR-8-11、WR-8-12、WR-8-13、WR-8-14、WR-8-15、WR-8-16、WR-
8-17 or WR-9.
In certain embodiments of the present invention, it is thermophilic the lipase that the lipase is generated by being selected from the group bacterium: to dredge cotton like
Hot hyphomycete (Thermomyces lanuginose), rhizomucor miehei (Rhizomucor miehei), rice black wool mould (Mucor
Miehei), pseudomonad (Pseudomonas sp.), head mold (Rhizopus sp.), aspergillus niger (Aspergillus
Niger), Burkholderia yielding lipase (Burkholderia sp.)；It is transformed by genetic engineering or protein engineering
The lipase obtained；Or commercially available lipase.
In some instances, the lipase be commercially available enzyme, as Novozymes Company of Denmark Lipozyme TL100L,
Palatase20000L。
In some instances, the immobilized lipase still keeps at least 80% after the wheel reaction of catalytic esterification 10
Catalysis activity.
In the second aspect of the present invention, a kind of carrier is provided.
The functional group of carrier provided by the invention is and C8-15Alkane end connection primary amine groups, the functional group
Content be 0.5~6mmol/g, pore-size distribution be 12~80nm.
In some instances, the functional group content of the carrier is 1~5mmol/g, and pore-size distribution is 15~60nm.
In some instances, the particle diameter distribution of the carrier is 0.1~0.4mm, preferably 0.15~0.3mm, specific surface
Product is 100~600m2/ g, preferably 150~500m2/g。
In some instances, the carrier is deacidite carrier.
In some examples of the invention, the skeleton of the carrier is polyacrylate or styrene type skeleton.
In other examples, the carrier is alkalescent acrylate ionomer exchanger resin or alkalescent polystyrene ion
Exchanger resin.
In other examples, the carrier is made of material selected from the group below: the cross-linked polymer of acrylate；Crosslinking
Polymethyl methacrylate；Methacrylate, dimethyl maleate, allyl acetate, acrylonitrile or methyl ethylene
The copolymer of ketone and styrene or divinylbenzene；Acrylate or methacrylate and ethyleneglycol dimethyacrylate
Cross-linked copolymer；Or the copolymer of styrene and divinylbenzene.
In other examples, the carrier is methyl acrylate-divinylbenzene copolymer or styrene and divinylbenzene
Copolymer.
In some instances, the deacidite is alkalescent acrylate ionomer exchanger resin, it is preferred that institute
State alkalescent acrylate ionomer exchanger resin to prepare using following methods: (i) preparation acrylic compounds-divinylbenzene is total to
Polymers；(ii) it is swollen the copolymer, and amination is carried out to it.
In other examples, the deacidite is by methyl acrylate (or hydroxy-ethyl acrylate, propylene
Sour polyglycidyl, methyl methacrylate, hydroxyethyl methacrylate, glycidyl methacrylate) and divinylbenzene with 20:
The mass ratio (preferably 15:1~10:1) of 1~1:1, pore-foaming agent (such as the mixture or gasoline of toluene and liquid wax, normal heptane,
Butyl acetate, 1,2- dichloroethanes) and initiator () benzoyl peroxide, azo-bis-isobutyl cyanide or potassium peroxydisulfate) existing
In the case of polymerize, the ratio of copolymer and pore-foaming agent therein is 3:1~1:3 (preferably 3:2~3:4), and initiator is above-mentioned body
0.1% (mass ratio) of system.The polymerization temperatures range of above-mentioned monomer is 60~100 DEG C, preferably 65~85 DEG C.Gained copolymer with
Sweller (or mixtures thereof N,N-dimethylformamide, nitrobenzene, methylene chloride, dichloroethanes, carbon disulfide, dioxane)
It is swollen with the volume ratio (preferably 1:2~1:10) of 1:1~1:15, swelling condition can are as follows: at room temperature, copolymer is in sweller
Middle immersion is swollen 4~24 hours, preferably 6~16 hours.By copolymer and 1,8- octamethylenediamine with methyl esters key and 1,8- in copolymer
The molar ratio of octamethylenediamine is with the ratio of 1:1~1:10 (preferably 1:2~1:5), under 100~150 DEG C (preferably 120~140 DEG C)
Amination, aminating reaction time are 4~24 hours (preferably 8~12 hours).
In some instances, the deacidite is alkalescent polystyrene ion-exchange resin, it is preferred that
The alkalescent polystyrene ion-exchange resin is prepared using following methods: (i') prepares polystyrene microsphere；(ii') it is swollen
The microballoon, and it is acylated that chlorine is carried out to it；(iii') it is swollen the chlorine acylated resin microballoon, and amination is carried out to it.
In some instances, the deacidite is by styrene, divinylbenzene and pore-foaming agent (e.g., toluene
With mixture or gasoline, normal heptane, butyl acetate, 1, the 2- dichloroethanes of liquid wax, preferably toluene and liquid wax 2:1 volume ratio
Mixture) in the presence of initiator (such as benzoyl peroxide, azo-bis-isobutyl cyanide, potassium peroxydisulfate, preferably benzoyl peroxide)
Under prepare polystyrene microsphere.Wherein, the mass ratio of styrene and divinylbenzene is 5:1~1:2, preferably 3:1~1:1；Benzene second
The mass ratio of alkene and divinylbenzene and pore-foaming agent is 1:1~1:6, preferably 1:1.5~1:4.The polymerization temperatures range of above-mentioned monomer
It is 60~95 DEG C, preferably 80~90 DEG C.Polymerization time range is 4~10 hours, preferably 6~8 hours.Gained polystyrene it is micro-
Ball (is preferably but not limited to methylene chloride, N, N- dimethyl formyl in 1:1~1:15 volume ratio, the preferably sweller of 1:2~1:10
Or mixtures thereof amine, nitrobenzene, methylene chloride, dichloroethanes, carbon disulfide, dioxane) in swelling.Swelling condition are as follows: room
Under temperature, copolymer impregnates in a solvent, is swollen 4~24 hours, preferably 6~16 hours.Benzal and 9- chlorine in above-mentioned microballoon
The molar ratio of pelargonyl chloride is 1:0.1~1:2, preferably 1:0.2~1:1, and 9- chlorine pelargonyl chloride and catalyst are (anhydrous zinc chloride, anhydrous
Stannic chloride, preferably anhydrous aluminum chloride) molar ratio be 1:0.1~1:2, preferably 1:0.2~1:1,30~75 DEG C (preferably 35~
50 DEG C) under carry out chlorine acylated, 2~12h of reaction time, preferably 3~8h.By resulting chlorine acylated resin microballoon in 1:1~1:15
Sweller (N,N-dimethylformamide, nitrobenzene, methylene chloride, dichloroethanes, two sulphur of volume ratio (preferably 1:2~1:10)
Change carbon, or mixtures thereof dioxane) in the presence of be swollen 4~10 hours, preferably 6~8 hours.With ammonium hydroxide, ethylenediamine, divinyl
Triamine, triethylene tetramine or the tetraethylenepentamine (mole and NH of chlorine in chlorine acylated resin3Mole ratio is excellent in 1:1~1:20
Select 1:8~1:15) at 40~80 DEG C, preferably 50~60 DEG C progress aminations, the aminating reaction time 4~24 hours, preferably 10~14
Hour.
In certain embodiments of the present invention, the functional group content of the carrier is 1~5mmol/g, pore-size distribution
For 15~60nm, particle diameter distribution is 0.15~0.3mm, and specific surface area is 150~500m2/g。
In some instances, the carrier is WR-8-1, WR-8-2, WR-8-3, WR-8-4, WR-8-5, WR-8-6, WR-
8-7、WR-8-8、WR-8-9、WR-8-10、WR-8-11、WR-8-12、WR-8-13、WR-8-14、WR-8-15、WR-8-16、WR-
8-17 or WR-9.
In the third aspect of the invention, a kind of method for preparing immobilized lipase of the invention, the side are provided
Method includes:
I) carrier is provided；
Ii) lipase is provided, and zymoprotein crosslinking agent is optionally added wherein；
Contact the carrier with the lipase, so that the lipase is fixed on the carrier；
Iv) optionally drying step iii) obtained in be fixed with the carrier of lipase, to obtain the immobilized lipase.
In certain embodiments of the present invention, further include carrying out following pretreatment to the carrier in step i): using
The lye and acid solution alternate immersion of 0.05mol/L~1mol/L rinse described carrier 1~5 time, then only with 0.05mol/L~
The lye of 1mol/L impregnates or flush vehicle 1 time again, is washed with water to water and is in neutrality.
In some instances, the acid in the pretreatment is selected from: HCl, H2SO4Or H3PO4, the alkali is selected from:
NaOH, ammonium hydroxide or KOH.
In other examples, the time of each lye and acid soak or flushing is 1~24 hour.
In other examples, the pretreatment are as follows: with carrier and lye or acid solution 1:1~1:5, preferably 1:1.5~1:
3, the w/v (w/v) of more preferable 1:2 impregnates or rinses the carrier, last after being repeated several times sequentially to be sour after first alkali
It with base extraction, is then washed with water to water and is in neutrality for one time.
In other examples, the zymoprotein crosslinking agent is selected from: glutaraldehyde solution, polyethyleneimine, polypropyleneimine,
Polyvinylamine, gelatin or spermine.
In certain embodiments of the present invention, step iii) described in carrier and the lipase ratio be 1:3~
3:1。
In some instances, make the processed load using shaking bath, gas bath shaking table, blender or magnetic stirring apparatus
Body is contacted and is fixed with lipase.
In other examples, the fixation degree of zymoprotein is judged by taking supernatant to survey residual protein content, it is described surplus
The measurement of remaining protein content is carried out using method selected from the group below: Coomassie Brilliant Blue, lorry method, biuret method, BCA method.
In other examples, when residual protein content is lower than 0.1mg/mL~0.5mL/mL, fixing step is terminated.
In other examples, the drying is carried out by method selected from the group below: be freeze-dried, be dried in vacuo,
Fluidized bed drying and drying at room temperature.
In the fourth aspect of the invention, a kind of method for carrying out transesterification, esterification, acidolysis or alcoholysis reaction is provided,
The method includes using immobilized lipase of the invention as catalyst.Fixation of the invention is also provided in present aspect
Change application of the lipase in catalytic transesterification, esterification, acidolysis and/or alcoholysis reaction.
In some instances, the method is applied to immobilized lipase of the invention while carrying out high Sn-1, and 3 specially
In the reaction of one property, catalytic transesterification and catalyzing esterification.
In other examples, the method or application are for being preferred for biodiesel, functionality in fats and oils processing industry
During grease, fine chemical product generate.
In other examples, the method or application are multiple batches of or continuous productions or reaction for taking turns raw materials more.
In in the fifth aspect of the invention, a kind of kit comprising immobilized lipase of the invention or production are provided
Product.In some instances, the kit or product further include packing material, preferably container or packaging bag.
In the sixth aspect of the present invention, additionally provides carrier as described above and preparing immobilized lipase of the invention
In application, the functional group of the carrier is and C8-15Alkane end connection primary amine groups, the functional group of the carrier
Content is 0.5~6mmol/g, and pore-size distribution is 12~80nm.
In some instances, the functional group content of the carrier is 1~5mmol/g, and pore-size distribution is 15~60nm.?
In some examples, the particle diameter distribution of the deacidite carrier is 0.1~0.4mm, preferably 0.15~0.3mm,
Specific surface area is 100~600m2/ g, preferably 150~500m2/g。
In some instances, the carrier is deacidite.
In some instances, the carrier is WR-8-1, WR-8-2, WR-8-3, WR-8-4, WR-8-5, WR-8-6, WR-
8-7、WR-8-8、WR-8-9、WR-8-10、WR-8-11、WR-8-12、WR-8-13、WR-8-14、WR-8-15、WR-8-16、WR-
8-17 or WR-9.
Other aspects of the invention are apparent to those skilled in the art due to this disclosure
's.
Specific embodiment
The present inventor is carried by long-term in-depth study using the deacidite with specific physical and chemical parameter
Carrier of the body as lipase, it is unexpected to be made while having both high ester exchange activity, strong Sn-1,3 specificities and high esterification
The immobilised enzymes of ability.On this basis, the present inventor completes the present invention.
1. the selection and pretreatment of carrier
The carrier (preferably deacidite carrier) with specific physical and chemical parameter is used in the present invention as solid
The carrier of immobilized lipase.As used herein, term " carrier of the invention ", which refers to, can carry other materials (such as lipase) altogether
With the constant substance for participating in certain chemically or physically process.As used herein, term " ion exchange resin " refers to functional
Group (active group for having exchange ion) has reticular structure, insoluble high-molecular compound, is commonly used for ion exchange layer
Analyse medium.
The functional group of carrier (preferably deacidite carrier) of the invention is and C8-15Alkane end connect
The primary amine groups connect, the functional group content of the carrier are 0.5~6mmol/g, preferably 1~5mmol/g, pore-size distribution 12
~80nm, preferably 15~60nm.In some instances, the particle diameter distribution of the carrier be 0.1~0.4mm, preferably 0.15
~0.3mm, specific surface area are 100~600m2/ g, preferably 150~500m2/g.In some embodiments, carrier of the present invention
Functional group content be 1~5mmol/g, pore-size distribution be 15~60nm, particle diameter distribution be 0.15~0.3mm, specific surface area
For 150~500m2/g。
The skeleton of carrier of the present invention can be polyacrylate or styrene type skeleton, can thus be alkaline acrylate ionomer
Exchanger resin or basic polystyrene ion exchange resin.The carrier can be made and (be not limited to) by material selected from the group below: third
The cross-linked polymer of olefin(e) acid ester；Crosslinked polymethylmethacrylaparticles；Methacrylate, dimethyl maleate, acetic acid alkene
The copolymer of propyl ester, acrylonitrile or methyl vinyl ketone and styrene or divinylbenzene；Acrylate or methacrylate
With the cross-linked copolymer of ethyleneglycol dimethyacrylate；Or the copolymer of styrene and divinylbenzene.Used carrier of the present invention
The preferably copolymer of methyl acrylate-divinylbenzene copolymer or styrene and divinylbenzene.Ion for use in the present invention
Exchanger resin includes but is not limited to: WR-8 series plastics (including such as WR-8-1, WR-8-2, WR-8-3, WR-8-4, WR-8-
5、WR-8-6、WR-8-7、WR-8-8、WR-8-9、WR-8-10、WR-8-11、WR-8-12、WR-8-13、WR-8-14、WR-8-
15, WR-8-16 and WR-8-17), WR-9 resin etc., or meet other resins of parameter area condition of the present invention.
It will be understood by those skilled in the art that the ion exchange resin carrier that can be used in the present invention with trade name in addition to being limited
It further include any commercial resins product with other trade names or by voluntarily preparing or processing other than fixed specific product
The resin arrived, also may be based on the modification or improved resin of above or other resins, such as pass through diazotising or amination modified
Resin or use other resins of the chemical modification means through functionalization, as long as they have the present invention defined by physics and chemistry
Parameter or with the same or similar structure of the specific product and absorption or switching performance.
Before using carrier immobilized enzyme of the invention, carrier can be pre-processed.Pretreated method include but
Be not limited to: with the lye (such as NaOH, KOH or ammonium hydroxide) of low concentration (0.05mol/L-1mol/L) and acid solution (such as HCl,
H2SO4Or H3PO4) (acid solution and the concentration of lye be 0.05mol/L-1mol/L) alternate treatment (processing method can be for for example:
1~3h of dipping by lye resin (preferably 2h) is first used, is then washed till neutrality, then with acid soak or after rinsing 1~3h (preferably 2h), wash
To neutrality, the process is repeated 1-5 times) resin 1-5 times, lye is only used in last time processing, and the time per treatment is 1-24 hours,
Water is washed till with water (preferably deionized water, distilled water or distilled water) after being disposed to be in neutrality, then is immersed in water (preferably deionization
Water, distilled water or distilled water) in save backup.PH inside ion exchange resin through above-mentioned processing remains alkalinity, more has
Its activity is played conducive to enzyme.
2. lipase and its immobilization
Lipase used can be generated by being selected from the group bacterium in the present invention: dredge the thermophilic hyphomycete (Thermomyces of cotton like
Lanuginose), rhizomucor miehei (Rhizomucor miehei), rice black wool mould (Mucor miehei), pseudomonad
(Pseudomonas sp.), head mold (Rhizopus sp.), aspergillus niger (Aspergillus niger), Burkholderia produce
Lipase (Burkholderia sp.)；It is repaired by lipase obtained by genetic engineering or protein engineering transformation, including chemistry
The enzyme that the enzyme of decorations, the mutant of protein engineering or genetic engineering bacterium generate；Or commercially available lipase, including but not limited to Denmark
Lipozyme TL100L, the Palatase20000L of Novozymes Company.
Any separation, purifying etc., which can be needed not move through, for the enzyme solution in immobilization process changes its original composition or environment
Pretreatment.Enzyme solution can also be diluted or pH adjust etc. processing, be suitable for reacting.
May include (but being not limited to) for TL enzyme solution of the invention:
1) by cultivating the fermentation liquid of thermophilic hyphomycete (Thermomyces Lanuginosus) acquisition (for example, see text
Offer Charlotte Pinholt, Mathias Fano, Charlotte Wiberg, Influence of glycosylation
on the adsorption of Thermomyces lanuginosus lipase to hydrophobic and
hydrophilic surfaces,European Journal of Pharmaceutical Sciences40(2010):273-
281)；
2) lipase gene is obtained by cultivating from thermophilic hyphomycete, is obtained with gene-modified aspergillus oryzae by fermenting and producing
The lipase enzyme solution obtained is (for example, see ibid document Charlotte Pinholt, Mathias Fano, Charlotte
Wiberg,European Journal of Pharmaceutical Sciences40(2010):273-281)。
3) commercially available commodity TL liquid aliphatic enzyme, such as the Lipozyme TL100L of Novozymes Company.
4) by the way that acid-base accommodation TL enzyme solution is added to suitable pH in the solution of fatty enzyme solution as described above or enzyme powder
Value and/or addition water (such as distilled water, deionized water or distilled water) adjust the enzyme solution to suitable protein concentration (for example, see text
Offer Daniel Otzen, Differential adsorption of variants of the Thermomyces
lanuginosus lipase on a hydrophobic surface suggests a role for local
Flexibility, biointerfaces200864:223-228).
Enzyme solution for its lipase in the present invention can also be used similar approach as described above and obtain.Such as it can be used
With aspergillus niger (Aspergillus niger) for the rhizomucor miehei lipase liquid (RM enzyme solution) of host expresses or with Pichia pastoris
For the RM enzyme solution of host expresses.
A certain amount of zymoprotein crosslinking agent can also be added in enzyme solution in the present invention (can play crosslinking role of apoenzyme
Substance), the preferably described zymoprotein crosslinking agent has hydroxyl or amino, such as glutaraldehyde solution, polyethyleneimine, polypropylene are sub-
Amine, polyvinylamine, gelatin or spermine etc..The dosage of the zymoprotein crosslinking agent is that 0.0~10% (v/ is added in every milliliter of enzyme solution
v).Such as the dosage of glutaraldehyde is 1% glutaraldehyde solution that 0.01~0.1mL is added in every milliliter of enzyme solution, and equal proportion can also be used
Other concentration glutaraldehyde solutions.
Weight (g)/volume (mL) of processed resin carrier and TL enzyme solution than that can be 25:1~1:25, preferably 10:1~
1:10, more preferable 1:1.
After obtaining carrier of the invention, absorption method or absorption+crosslinking combined method can be used usually to realize enzyme
Immobilization.It can first activated carrier, then immobilized enzyme as needed；Or first activating enzymes, then the enzyme of activation is fixed on carrier.
In certain embodiments of the present invention, lipase is fixed on carrier of the invention using absorption method.It can be such as
The lower absorption for carrying out enzyme is fixed: being weighed a certain amount of pretreated carrier in container, the TL enzyme solution of certain volume is put into, in 15
~35 DEG C (preferably 15~25 DEG C, more preferable room temperature) are shaken (as stirred using shaking bath, gas bath shaking table, mechanical agitator, magnetic force
Mix device, vortex stirrer etc.) it is fixed, shaking speed can be such as 10~120rpm (such as 150rpm).After the completion of fixation, take out
It is adsorbed with the carrier of enzyme, and removes residual liquid.
During fixation, (such as 5min~1h) supernatant can be taken to survey residual protein content to judge zymoprotein at regular intervals
Absorption/combination degree.Coomassie Brilliant Blue, Lorry method, biuret method, BCA method etc. can be used in the measurement of residual protein content
Conventional protein quantitative detecting method carries out.In general, being terminated solid when residual protein content is lower than 0.1mg/mL~0.5mL/mL
Determine step.Certainly, those of ordinary skill in the art can according to specific needs (such as quality control requirement) to fixation degree into
Row adjustment and control.
3. dry
After the completion of immobilization step, it can further pass through moisture extra in drying steps removal system.The step
It can prevent undried immobilised enzymes from introducing water and leading to that other side reactions occur.Dry mode can be selected from: freeze-drying,
The conventional drying methods such as vacuum drying, fluidized bed drying and drying at room temperature.
For example, freeze-drying can LABCNCO freeze drier (manufacturer: Labcnco company of the U.S., model:
Freezone it is carried out on), parameter may be configured as (being each step parameter below, these steps constitute a temperature-fall period):
- 20 DEG C of rate of temperature fall 1.5 DEG C/min, 0.5-5h (such as 2h),
- 10 DEG C of rate of temperature fall 1.5 DEG C/min, 3-10h (such as 6h)
5 DEG C of rate of temperature fall 1.01 DEG C/min, 10-24h (such as 15h)
20 DEG C of rate of temperature fall 1.16 DEG C/min, 10-24h (such as 20h)
Vacuum drying can such as Binder vacuum oven (manufacturer: guest's moral Asia-Pacific (Hong Kong) Co., Ltd model:
VD23 it is carried out in), parameter is set as in the case where vacuum degree is 6mbar, 40 DEG C of dry 24-60h (such as 48h).
Fluidized bed drying can carry out (such as Gera, Germany spy company, model: midi glatt) in a fluidized bed, and parameter is set
It is set to 45~55 DEG C of inlet air temperature (preferably 50 DEG C), drying time is 0.5~5h (preferably 1.5h).
Drying at room temperature can be placed 3~5 days for the place that sample is placed on aeration-drying and be spontaneously dried.
Those of ordinary skill in the art can as needed select conventional drying methods with condition.
4. other optional steps
According to specific needs, can also additionally have following one or more optional steps, example in method of the invention
Such as (but not limited to):
1 is removed by filtration the small molecule in enzyme solution, to eliminate influence of the enzyme solution small molecular to enzyme activity.Filter method can
For for example: carrying out ultrafiltration (manufacturer: Millipore Corp. of the U.S., model: small-sized) in ultrafiltration diaphragm, phosphate buffer is then added
(such as pH be 5 0.1mol/L phosphate buffer), so that gained enzyme solution is consistent with protoenzyme liquid system pH, and be adjusted to
Zymoprotein concentration appropriate.
2 can be added the stability of the substances such as dextrin, albumin and/or trehalose enhancing enzyme in immobilization step, it
Respective preferred additive amount are as follows: dextrin 1~20% (w/v)；Albumin 0.05~5% (w/v)；Trehalose 0.1-10% (w/v).
One or more above-mentioned substances can be added, to obtain required stablizing effect.
3 pairs of carriers are modified processing, connect active group by chemical means.For example, can refer to Xu Jingliang etc., amino
Function carrier Research Progress of Immobilized Enzyme, chemical industry progress, 2010,29 (3): 494-496.
5. the preferred embodiment of the present invention
An exemplary preferred embodiment of the invention is as follows:
1) pretreatment of carrier
With the lye (such as NaOH, KOH) and acid solution of low concentration (0.05mol/L-1mol/L) (such as HCl, H2SO4Or
H3PO4) (processing method can be for for example: first (preferably with 1~3h of dipping by lye resin for (0.05mol/L-1mol/L) alternate treatment
2h), it is then washed till neutrality, then with acid soak or after rinsing 1~3h (preferably 2h), is washed till neutrality, repeats the process 1-5 times) tree
Rouge 1-5 times, last time processing are lye, and the time per treatment is 1-24 hours, and water (preferably deionization is used after being disposed
Water, distilled water or distilled water) it is washed till water and is in neutrality, then be immersed in water (preferably deionized water, distilled water or distilled water) and save
It is spare.
2) enzyme immobilizatio
Carrier and enzyme solution are placed in container according to a certain percentage, certain revolving speed is fixed in constant temperature air bath shaker,
Carrier can be handled as needed before immobilization.Period takes supernatant to survey residual protein content to judge at regular intervals
The degree of absorption of zymoprotein.
3) drying of immobilised enzymes
Extra moisture, that is, drying in the system that go out after immobilization step, drying mode can be to be placed on nature to lead to
The place of wind spontaneously dries, can also be dry with vacuum freezedrying or fluidized bed process.
It should be understood that above-mentioned preferred embodiment is only exemplary, those of ordinary skill in the art are based in the application
Record and the common sense of this field can without departing from the spirit and scope of the present invention, each Step By Condition is changed and
Optimization.
6. the application of immobilized lipase of the present invention
Immobilized lipase of the invention can be directly used in industrialized production, can also be made into packaging product so as to
In being stored, transported, sold and further used.
It is preferably filling in clean container under conditions of sterile dry low temperature when packaging.Storage condition is preferably low
Warm drying condition storage, storage optimum temperature are 4~20 DEG C.It is preferred that 4~25 DEG C at a temperature of transported.
Immobilized lipase of the invention or its pack product can catalytic transesterification and esterification simultaneously, and also have
Stronger Sn-1,3 selectivity, can be widely applied on the fields such as food, papermaking, leather, detergent, pharmacy, is preferred for grease
In processing industry, in biodiesel, functional grease, fine chemical product production, it can be used for being catalyzed grease and hydrolysis, ester friendship occur
It changes, esterification or catalysis acidolysis, alcoholysis etc..
7. advantages of the present invention
1) using a kind of novel immobilized lipase is provided in the present invention, which has catalysis simultaneously
Esterification, the function of ester exchange reaction and high Sn-1,3 selectivity, strong tool is provided for industrial production；
2) the present invention provides a kind of new method for preparing immobilized lipase, using in this method has specific physics and chemistry
The ion exchange resin of parameter achieves good fixed effect as fixation support, and obtains and have the above advantages
Immobilized lipase, this method is not necessarily to carry out lipase complicated pretreatment, to enormously simplify production stage, reduce
Production cost.
3) immobilized lipase of the invention can be used for successive reaction, and can recycle, and be produced into reduce
This, improves utilization rate and speed of production.
Embodiment
Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention
Rather than it limits the scope of the invention.Those skilled in the art can make modification appropriate, variation to the present invention, these modifications
It is within the scope of the present invention with variation.In the following examples, the experimental methods for specific conditions are not specified, usually according to routine
Condition condition as described in " Biochemistry and Molecular Biology experiment textbook " (Liang Songping edits Higher Education Publishing House), or
According to the normal condition proposed by manufacturer.Unless otherwise stated, otherwise percentage and number are calculated by weight.
Unless otherwise defined, it anticipates known to all professional and scientific terms as used herein and one skilled in the art
Justice is identical.In addition, any method similar to or equal to what is recorded and material can be applied to the method for the present invention.Wen Zhong
The preferred implement methods and materials are for illustrative purposes only.
The preparation of 1. immobilised enzymes of embodiment
The method for preparing immobilised enzymes is present embodiments provided, specific as follows:
(A) synthetic method of immobilised enzymes
(a) 50mL commodity TL enzyme solution (Lipozyme TL100L, Novozymes Company) preparation of TL enzyme solution: is placed in freezing
It is freeze-dried in drying machine.Freeze-drying can be in freeze drier (manufacturer: Labcnco company of the U.S., model: freezone)
It carries out, parameter setting are as follows:
- 15 DEG C, rate of temperature fall 1.2 DEG C/min, 0.5~3h (such as 2h),
- 10 DEG C, rate of temperature fall 1.5 DEG C/min, 3~10h (such as 5h),
5 DEG C, rate of temperature fall 1.5 DEG C/min, 10~20h (such as 12h),
20 DEG C, rate of temperature fall 1.5 DEG C/min, 10~20h (such as 15h).
The pH7 phosphate buffer 50mL redissolution that 100mmol/L is added in the resulting sample of drying is spare.
(b) it is dry that 50mL commodity RM enzyme solution (Palatase20000L Novozymes Company) preparation of RM enzyme solution: is placed in freezing
It is freeze-dried in dry machine.Freeze-drying can be enterprising in freeze drier (manufacturer: Labcnco company of the U.S., model: freezone)
Row, parameter setting are as follows:
- 15 DEG C, rate of temperature fall 1.2 DEG C/min, 0.5~3h (such as 2h),
- 10 DEG C, rate of temperature fall 1.5 DEG C/min, 3~10h (such as 5h),
5 DEG C, rate of temperature fall 1.5 DEG C/min, 10~20h (such as 12h),
20 DEG C, rate of temperature fall 1.5 DEG C/min, 10~20h (such as 15h).
The pH7 phosphate buffer 50mL redissolution that 100mmol/L is added in the resulting enzyme powder of drying is spare.
(c) measuring method of resin parameter
Aperture, specific surface area index determining use Bei Shide (model 3H-2000PS1) nitrogen adsorption instrument；Particle size determination
Using Beckman (model LS13320) laser particle analyzer；Functional group content is measured according to national standard GB/T19861-2005.
1. immobilization TL enzyme (absorption method)
Weigh resin (its functional group content 1.0mmol/g, average pore size 30nm, the average specific that 15g number is WR-8-1
Surface area 250m2/ g, average grain diameter 0.22mm), it is placed in 100mL round-bottomed flask, 10mL TL enzyme solution (Lipozyme is added
TL100L, Novozymes company), 150rpm vibrates 4h in 25 DEG C of shaking tables, and enzyme taking-up is then put into clean culture dish
In, it is put into draught cupboard and is dried, obtain immobilised enzymes 1 after dry.
2. immobilization TL enzyme (absorption method)
Weigh resin (functional group content 2.5mmol/g, average pore size 30nm, the average specific surface that 15g number is WR-9
Product 250m2/ g, average grain diameter 0.22mm), it is placed in 100mL round-bottomed flask, addition 10mLTL enzyme solution (Lipozyme TL100L,
Novozymes company), 150rpm vibrates 4h in 25 DEG C of shaking tables, and then enzyme taking-up is put into clean culture dish, is put into logical
It is dried in wind cupboard, obtains immobilised enzymes 2 after dry.
3. immobilization TL enzyme (absorption method)
Weigh resin (functional group content 2.5mmol/g, average pore size 30nm, the average specific table that 15g number is WR-8-2
Area 250m2/ g, average grain diameter 0.22mm), it is placed in 100mL round-bottomed flask, 10mL TL enzyme solution (Lipozyme is added
TL100L, Novozymes company), 150rpm vibrates 4h in 25 DEG C of shaking tables, and enzyme taking-up is then put into clean culture dish
In, it is put into draught cupboard and is dried, obtain immobilised enzymes 3 after dry.
4. immobilization TL enzyme (absorption method)
Weigh resin (functional group content 5mmol/g, average pore size 30nm, the average specific surface that 15g number is WR-8-3
Product 250m2/ g, average grain diameter 0.22mm), it is placed in 100mL round-bottomed flask, 10mL TL enzyme solution (Lipozyme is added
TL100L, Novozymes company), 150rpm vibrates 4h in 25 DEG C of shaking tables, and enzyme taking-up is then put into clean culture dish
In, it is put into draught cupboard and is dried, obtain immobilised enzymes 4 after dry.
5. immobilization TL enzyme (absorption method)
Weigh resin (functional group content 2.5mmol/g, average pore size 15nm, the average specific table that 15g number is WR-8-4
Area 250m2/ g, average grain diameter 0.22mm), it is placed in 100mL round-bottomed flask, 10mL TL enzyme solution (Lipozyme is added
TL100L, Novozymes company), 150rpm vibrates 4h in 25 DEG C of shaking tables, and enzyme taking-up is then put into clean culture dish
In, it is put into draught cupboard and is dried, obtain immobilised enzymes 5 after dry.
6. immobilization TL enzyme (absorption method)
Weigh resin (functional group content 2.5mmol/g, average pore size 60nm, the average specific table that 15g number is WR-8-5
Area 250m2/ g, average grain diameter 0.22mm), it is placed in 100mL round-bottomed flask, 10mL TL enzyme solution (Lipozyme is added
TL100L, Novozymes company), 150rpm vibrates 4h in 25 DEG C of shaking tables, and enzyme taking-up is then put into clean culture dish
In, it is put into draught cupboard and is dried, obtain immobilised enzymes 6 after dry.
7. immobilization TL enzyme (absorption method)
Weigh resin (functional group content 2.5mmol/g, average pore size 30nm, the average specific table that 15g number is WR-8-6
Area 150m2/ g, average grain diameter 0.22mm), it is placed in 100mL round-bottomed flask, 10mL TL enzyme solution (Lipozyme is added
TL100L, Novozymes company), 150rpm vibrates 4h in 25 DEG C of shaking tables, and enzyme taking-up is then put into clean culture dish
In, it is put into draught cupboard and is dried, obtain immobilised enzymes 7 after dry.
8. immobilization TL enzyme (absorption method)
Weigh resin (functional group content 2.5mmol/g, average pore size 30nm, the average specific table that 15g number is WR-8-7
Area 500m2/ g, average grain diameter 0.22mm), it is placed in 100mL round-bottomed flask, 10mL TL enzyme solution (Lipozyme is added
TL100L, Novozymes company), 150rpm vibrates 4h in 25 DEG C of shaking tables, and enzyme taking-up is then put into clean culture dish
In, it is put into draught cupboard and is dried, obtain immobilised enzymes 8 after dry.
9. immobilization TL enzyme (absorption method)
Weigh resin (functional group content 2.5mmol/g, average pore size 30nm, the average specific table that 15g number is WR-8-8
Area 250m2/ g, average grain diameter 0.15mm), it is placed in 100mL round-bottomed flask, 10mL TL enzyme solution (Lipozyme is added
TL100L, Novozymes company), 150rpm vibrates 4h in 25 DEG C of shaking tables, and enzyme taking-up is then put into clean culture dish
In, it is put into draught cupboard and is dried, obtain immobilised enzymes 9 after dry.
10. immobilization TL enzyme (absorption method)
Weigh resin (functional group content 2.5mmol/g, average pore size 30nm, the average specific table that 15g number is WR-8-9
Area 250m2/ g, average grain diameter 0.3mm), it is placed in 100mL round-bottomed flask, 10mLTL enzyme solution (Lipozyme is added
TL100L, Novozymes company), 150rpm vibrates 4h in 25 DEG C of shaking tables, and enzyme taking-up is then put into clean culture dish
In, it is put into draught cupboard and is dried, obtain immobilised enzymes 10 after dry.
11. immobilization TL enzyme (absorption method)
Weigh resin (functional group content 0.2mmol/g, average pore size 30nm, the average specific that 15g number is WR-8-10
Surface area 250m2/ g, average grain diameter 0.22mm), it is placed in 100mL round-bottomed flask, 10mL TL enzyme solution (Lipozyme is added
TL100L, Novozymes company), 150rpm vibrates 4h in 25 DEG C of shaking tables, and enzyme taking-up is then put into clean culture dish
In, it is put into draught cupboard and is dried, immobilised enzymes 11 must be compared after dry.
12. immobilization TL enzyme (absorption method)
Weigh resin (functional group content 7.0mmol/g, average pore size 30nm, the average specific that 15g number is WR-8-11
Surface area 250m2/ g, average grain diameter 0.22mm), it is placed in 100mL round-bottomed flask, 10mL TL enzyme solution (Lipozyme is added
TL100L, Novozymes company), 150rpm vibrates 4h in 25 DEG C of shaking tables, and enzyme taking-up is then put into clean culture dish
In, it is put into draught cupboard and is dried, immobilised enzymes 12 must be compared after dry.
13. immobilization TL enzyme (absorption method)
Weigh resin (functional group content 2.5mmol/g, average pore size 5nm, the average specific table that 15g number is WR-8-12
Area 250m2/ g, average grain diameter 0.22mm), it is placed in 100mL round-bottomed flask, 10mL TL enzyme solution (Lipozyme is added
TL100L, Novozymes company), 150rpm vibrates 4h in 25 DEG C of shaking tables, and enzyme taking-up is then put into clean culture dish
In, it is put into draught cupboard and is dried, immobilised enzymes 13 must be compared after dry.
14. immobilization TL enzyme (absorption method)
Weigh resin (functional group content 2.5mmol/g, average pore size 200nm, the average specific that 15g number is WR-8-13
Surface area 250m2/ g, average grain diameter 0.22mm), it is placed in 100mL round-bottomed flask, 10mL TL enzyme solution (Lipozyme is added
TL100L, Novozymes company), 150rpm vibrates 4h in 25 DEG C of shaking tables, and enzyme taking-up is then put into clean culture dish
In, it is put into draught cupboard and is dried, immobilised enzymes 14 must be compared after dry.
15. immobilization TL enzyme (absorption method)
Weigh resin (functional group content 2.5mmol/g, average pore size 30nm, the average specific that 15g number is WR-8-14
Surface area 50m2/ g, average grain diameter 0.22mm), it is placed in 100mL round-bottomed flask, 10mL TL enzyme solution (Lipozyme is added
TL100L, Novozymes company), 150rpm vibrates 4h in 25 DEG C of shaking tables, and enzyme taking-up is then put into clean culture dish
In, it is put into draught cupboard and is dried, immobilised enzymes 15 must be compared after dry.
16. immobilization TL enzyme (absorption method)
Weigh resin (functional group content 2.5mmol/g, average pore size 30nm, the average specific that 15g number is WR-8-15
Surface area 750m2/ g, average grain diameter 0.22mm), it is placed in 100mL round-bottomed flask, 10mL TL enzyme solution (Lipozyme is added
TL100L, Novozymes company), 150rpm vibrates 4h in 25 DEG C of shaking tables, and enzyme taking-up is then put into clean culture dish
In, it is put into draught cupboard and is dried, immobilised enzymes 16 must be compared after dry.
17. immobilization TL enzyme (absorption method)
Weigh resin (functional group content 2.5mmol/g, average pore size 30nm, the average specific that 15g number is WR-8-16
Surface area 250m2/ g, average grain diameter 0.05mm), it is placed in 100mL round-bottomed flask, 10mL TL enzyme solution (Lipozyme is added
TL100L, Novozymes company), 150rpm vibrates 4h in 25 DEG C of shaking tables, and enzyme taking-up is then put into clean culture dish
In, it is put into draught cupboard and is dried, immobilised enzymes 17 must be compared after dry.
18. immobilization TL enzyme (absorption method)
Weigh resin (functional group content 2.5mmol/g, average pore size 30nm, the average specific that 15g number is WR-8-17
Surface area 250m2/ g, average grain diameter 0.5mm), it is placed in 100mL round-bottomed flask, 10mL TL enzyme solution (Lipozyme is added
TL100L, Novozymes company), 150rpm vibrates 4h in 25 DEG C of shaking tables, and enzyme taking-up is then put into clean culture dish
In, it is put into draught cupboard and is dried, immobilised enzymes 18 must be compared after dry.
19. immobilization TL enzyme (absorption method)
Weighing the resin that 15g number is D311 (Anhui Samsung resin processing plant), (functional group is directly to connect with resin matrix
Primaquine group, functional group content be 2.5mmol/g, average pore size 100nm, average specific surface area 250m2/ g, average grain diameter
0.6mm), it is placed in 100mL round-bottomed flask, is added 10mL TL enzyme solution (Lipozyme TL100L, Novozymes company),
150rpm vibrates 4h in 25 DEG C of shaking tables, and then enzyme taking-up is put into clean culture dish, is put into draught cupboard and is dried, is done
Immobilised enzymes 19 must be compared after dry.
20. immobilization RM enzyme (absorption method)
Weigh resin (functional group content 2.5mmol/g, average pore size 30nm, the average specific table that 10g number is WR-8-2
Area 250m2/ g, average grain diameter 0.22mm), it is placed in 100mL round-bottomed flask, 20mLRM enzyme solution is added (to be purchased from
The Pichia pastoris of invitrogen company is the RM gene that host strain expression GenBank accession number is ABI17884.1, acquisition
Enzyme solution；Preparation method reference: Li Huazhen etc., " Pichia pastoris surface display rhizomucor miehei lipase fermentation condition "；Food hair
Ferment industry, 2011, vol.37, No.9), 150rpm vibrates 2h in 25 DEG C of shaking tables, and enzyme taking-up is then put into clean culture
It in ware, is put into draught cupboard and is dried, obtain immobilised enzymes 20 after dry.
21. immobilization RM enzyme (absorption method)
Weigh resin (functional group content 2.5mmol/g, average pore size 30nm, the average specific table that 10g number is WR-8-2
Area 250m2/ g, average grain diameter 0.22mm), it is placed in 100mL round-bottomed flask, 20mLRM enzyme solution is added (to be purchased from
The aspergillus niger (Aspergillus niger) of invitrogen company is that host strain expression GenBank accession number is
The RM gene of ABI17884.1, the RM enzyme solution of acquisition；Preparation method reference: Cui Cui, " rhizomucor miehei lipase gene is in aspergillus
In expression study ", South China Science & Engineering University's Master's thesis), in 25 DEG C of shaking tables 150rpm vibrate 2h, then by enzyme taking-up be put into
It in clean culture dish, is put into draught cupboard and is dried, obtain immobilised enzymes 21 after dry.
22. immobilization RM enzyme (absorption method)
Weigh resin (functional group content 2.5mmol/g, average pore size 30nm, the average specific table that 10g number is WR-8-2
Area 250m2/ g, average grain diameter 0.22mm), it is placed in 100mL round-bottomed flask, 20mL aspergillus oryzae (Aspergillus is added
Oryzae) be host strain expression (RM gene is the RM gene that Genebank accession number be ABI17884.1) RM enzyme solution (prepare
Method reference: Cui Cui, " expression study of the rhizomucor miehei lipase gene in aspergillus ", South China Science & Engineering University's Master's thesis),
150rpm vibrates 2h in 25 DEG C of shaking tables, and then enzyme taking-up is put into clean culture dish, is put into draught cupboard and is dried,
Immobilised enzymes 22 is obtained after drying.
(B) synthetic method of WR-8 resin
At 45 DEG C, methyl acrylate, divinylbenzene, gasoline, (200# gasoline is purchased from the limited public affairs of traditional Chinese medicines chemical reagent
Department) it is uniformly mixed, the benzoyl peroxide of above-mentioned volume 0.1% is added as initiator, is uniformly mixed.It is added to dissolved with gelatin
In the 200mL deionized water of (concentration is 0.1% weight ratio), saturated sodium-chloride, start to react after stirring is warming up to 75~80 DEG C
6h keeps the temperature 4~6h at 75-85 DEG C, then pumps mother liquor, washes, and after dry, obtains methyl acrylate-divinylbenzene copolymerization
Object.Then, 10g copolymer is swollen in 100mLN, dinethylformamide 2h, a certain amount of 1,8- octamethylenediamine (propylene is added
In sour methyl esters divinylbenzene copolymer the molar ratio of the mole of methyl esters key and 1,8- octamethylenediamine the preferred 1:2 of 1:1~1:10~
Aminating reaction 1:5) is carried out, 120~140 DEG C of (temperature conditions of the aminating reaction of every kind of resin of WR-8-1~WR-8-17 are warming up to
Difference) after, isothermal reaction for a period of time, then pumps mother liquor, is washed with deionized, and filters, obtains alkalescent propylene
Acid ion exchange.It is detected through national standard GB/T19861-2005, the functional group of WR-8-1~WR-8-17 is the alkane of 8 carbon
Hydrocarbon end connects a primary amine group, and concrete content is as shown in table 2.
The synthetic parameters of WR-8-1~WR-8-17 are as described in Table 1.
(C) synthetic method of WR-9 resin
Styrene, divinylbenzene, perforating agent (toluene: liquid wax=2:1 volume ratio) are pre-mixed uniformly under room temperature state
(mixed proportion is 3:2:10 mass ratio), 0.2g initiator benzoyl peroxide is added in 70g said mixture, is added to molten
There is 500mL in the pure water of dispersing agent gelatin (concentration is 0.1% weight percent), stirs and be segmented, 78 DEG C of heat preservation 4h are warming up to
95 DEG C, 6h is kept the temperature, mother liquor is then pumped, is washed 3~4 times with 90 DEG C of hot pure waters.Clean product is after boiled bed drying screens
The resin that finished product polystyrene is skeleton can be obtained.
The above-mentioned resin of 10g is added in there-necked flask, adds 100mL methylene chloride swelling 6h, and stirring keeps its dispersion equal
It is even, it is added dropwise 6g acylting agent (9- chlorine pelargonyl chloride), is slowly added under stirring 5g catalyst (aluminum trichloride (anhydrous)), it is anti-under room temperature
4h between seasonable.After reaction, pump mother liquor, be washed with deionized, filter, after dry, then plus 100mL N, N- dimethyl
Formamide swelling overnight, is added 26mL28% ammonium hydroxide, is warming up to 60 DEG C and is reacted.After reacting 12h, mother liquor is pumped, is spent
Alkalescent polystyrene ion-exchange resin is obtained by filtration in ion water washing.
It being detected through national standard GB/T19861-2005, functional group is that the alkane end of 8 carbon connects a primary amine group,
Content is 2.5mmol/g.
Adsorption rate is tested using Bradford method.As the result is shown: each carrier is to the adsorption rate of enzyme solution 90% in this method
More than.
Using WR-8 serial carrier, WR-9 carrier and commercially available D311 carrier in this present embodiment physical property and its
Application in immobilised enzymes preparation in the present embodiment is summarized in table 2.
The synthetic parameters of table 1.WR-8-1~WR-8-17
2. immobilised enzymes of table and its used carrier property are summarized
The measurement of 2. immobilised enzymes ester exchange activity of embodiment
It is (limited purchased from special oil in praising using purified soyabean oil (commercially available ingot board soybean oil) and deep hydrogenation soybean oil
Company, lot number LHS68-03) it is substrate (w/w73:37), enzyme (in terms of the gross mass of carrier and enzyme) and substrate mass ratio are 1:
20, in 70 DEG C of reaction 30min, reaction takes out product after finishing and surveys its 40 DEG C of solid fats content (SFC) contents.Product is taken out every time
Enzyme is stayed in reactor, substrate is added and is reacted, carries out 5 wheel reactions.
40 DEG C of SFC content methods: the solid rouge pipe equipped with sample is respectively put into 15min in 100 DEG C of baking ovens, 60 DEG C of water-baths
Then 5min, 0 DEG C of water-bath 60min, 40 DEG C of water-bath 30min survey its solid rouge content with Nuclear Magnetic Resonance.
The calculation formula of ester exchange activity is as follows:
Ester exchange activity=(SFCBlank- SFCSample)/30×1260
Wherein, SFCBlankRefer to the SFC value at 40 DEG C of the substrate grease not Jing Guo enzyme process ester exchange reaction,
SFCSampleRefer to the SFC value of enzyme reaction product sample at 40 DEG C.
Experimental result is the average value of 3 parallel laboratory tests.
Ester exchange activity measurement result is as shown in table 3.
From the results, it was seen that selecting specific support (functional group content: 1~5mmol/g in the present invention；Pore-size distribution:
15~60nm；Particle diameter distribution: 0.15~0.3mm；Specific surface area: 150~500m2/ g) fixation prepared is combined with different enzyme solutions
Change enzyme ester exchange activity be higher than or be even significantly higher than corresponding commodity immobilised enzymes Lipozyme TL IM or
Lipozyme RM IM。
Using immobilised enzymes 11-18 as comparative example, by data as can be seen that working as aperture, specific surface area, partial size and/or function
When in range of the energy group content not specified by the present invention, the transesterification effect of examples of such carriers immobilised enzymes is poor.Immobilization
Using commercialization acrylic acid weak base carrier D311 in enzyme 19, from data as can be seen that the functional group when carrier is different from
When the group used in carrier of the present invention, the transesterification effect of immobilised enzymes is also poor.
Therefore, it is obtained this example demonstrated the present invention by the combination of specific support and enzyme and is imitated with excellent transesterification
The immobilised enzymes of rate.
The measurement result of 3. immobilised enzymes ester exchange activity of table
The investigation of 3. immobilised enzymes specificity of embodiment
25g palmitinic acid stearin and 50g oleic acid are added in 250mL triangular flask, 5g immobilised enzymes to be measured is separately added into, in
60 DEG C, 150rpm reacts 3.5h.After reaction take out product be reentered into substrate reacted (i.e. every time take out product enzyme is stayed in
In reactor, add substrate and reacted), carry out 4 wheel reactions).The reaction of immobilized enzyme catalysis is shown below (wherein p
Indicate that palmitic acid moieties, o indicate oleic acid moiety):
The analysis of reaction product: after completion of the reaction, with gas chromatograph, (instrument model Agilent Agilent7890A is tested
Condition: 250 DEG C of inlet temperature, H2Flow 40mL/min, split ratio 75:1) carry out FAC (fatty acid composition), Sn-2 and TAG it is (sweet
Three esters composition) analysis.
The insertion rate calculation formula of 2 p (i.e. palmitinic acid) are as follows:
Sn-2 palmitinic acids account for ratio=Sn-2 palmitinic acid content of fatty acid × 100% of total palmitinic acid/(3 × total palm
Acid content)
Result shown in table 4 is that the immobilised enzymes that is obtained using the method for the present invention is anti-in the acidolysis of palmitinic acid stearin and oleic acid
The comparison of 2 insertion rates of product after answering.
What 2 P insertion rates reflected is the specificity of enzyme, and insertion rate is higher, and the specificity of enzyme is stronger.
From experimental result as can be seen that selecting specific support (functional group content: 1~5mmol/g in the present invention；Aperture
Distribution: 15~60nm；Particle diameter distribution: 0.15~0.3mm；Specific surface area: 150~500m2/ g) preparation is combined with different enzyme solutions
The specificity of immobilised enzymes is above corresponding commodity immobilised enzymes Lipozyme TL IM or Lipozyme RM IM.
Using immobilised enzymes 11-18 as comparative example, by data as can be seen that working as aperture, specific surface area, partial size and/or function
When in range of the energy group content not specified by the present invention, the specificity of examples of such carriers immobilised enzymes is poor.Immobilised enzymes 19
In using commercialization acrylic acid weak base carrier D311, from data as can be seen that when carrier functional group be different from this hair
When functional group used in bright carrier, the specificity of immobilised enzymes is also poor.
Therefore, this example demonstrated the present invention is obtained by the combination of specific support and enzyme with excellent specificity
Immobilised enzymes.
The investigation result of 4. immobilised enzymes specificity of table
The investigation of 4. immobilised enzymes of embodiment esterification ability
28g PFAD (Palm Fatty Acid Distillation, palm fibre is added into 100mL single necked round bottom flask respectively
Palmitic acid acid deodorization distillate), the various immobilised enzymes of 3g are placed on magnetic stirring apparatus 50 DEG C, 250rpm heating.Take methanol 3.2g, 6h
It is inside added in six times, takes out product after completion of the reaction, its methyl esters is analyzed by thin-layer chromatography-flame ion detection (TLC-FID)
Content, enzyme repeat above-mentioned reaction 10 times (take out product every time to stay in reactor enzyme, add substrate and reacted, into
The wheel of row 10 reaction).
More resulting esterification yield, i.e., the content of the ester generated in acid and alcohol reaction product.Esterification yield calculation formula are as follows:
Esterification yield=(100- product acid value/2)/100 × 100%
The results are shown in Table 5.
From the results, it was seen that selecting specific support (functional group content: 1~5mmol/g in the present invention；Pore-size distribution:
15~60nm；Particle diameter distribution: 0.15~0.3mm；Specific surface area: 150~500m2/ g) fixation prepared is combined with different enzyme solutions
The esterification ability for changing enzyme is preferable.Also, also there is 80% or so esterification activity after 10 wheel of immobilised enzymes reaction of the invention, it is bright
It is aobvious (not have ester substantially after several wheels reactions higher than corresponding commodity immobilised enzymes Lipozyme TL IM or Lipozyme RM IM
Change vigor).
Using immobilised enzymes 11-18 as comparative example, by data as can be seen that working as aperture, specific surface area, partial size and/or function
When in range of the energy group content not specified by the present invention, the esterification ability of examples of such carriers immobilised enzymes is poor, and in several wheels
Substantially do not have esterification activity after reaction.Using commercialization acrylic acid weak base carrier D311 in immobilised enzymes 19, from data
As can be seen that when the functional group of carrier is different from the group used in carrier of the present invention (alkane end connects primary amine group)
When, the esterification ability of immobilised enzymes is also poor, and does not have esterification activity substantially after the reaction of several wheels.
Therefore, this example demonstrated the present invention is obtained by the combination of specific support and enzyme with excellent and lasting ester
The immobilised enzymes of change ability.
The investigation result of 5. immobilised enzymes of table esterification ability
All references mentioned in the present invention is incorporated herein by reference, independent just as each document
It is incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can
To make various changes or modifications to the present invention, such equivalent forms equally fall within model defined by the application the appended claims
It encloses.
Claims (10)
1. a kind of immobilized lipase, it includes:
(a) carrier, wherein the functional group of the carrier is and C8-15Alkane end connection primary amine groups；
(b) it is fixed on the lipase of the carrier,
Wherein, the carrier is deacidite carrier, and wherein, the functional group content of the carrier is 1~
5mmol/g, pore-size distribution are 15~60nm, and particle diameter distribution is 0.15~0.3mm, and specific surface area is 150~500m2/g。
2. immobilized lipase as described in claim 1, wherein the skeleton of the carrier is polyacrylate or styrene
Type skeleton.
3. immobilized lipase as described in claim 1, which is characterized in that the rouge that the lipase is generated by being selected from the group bacterium
Fat enzyme: thin the thermophilic hyphomycete of cotton like (Thermomyces lanuginose), rhizomucor miehei (Rhizomucor miehei),
Rice black wool mould (Mucor miehei), pseudomonad (Pseudomonas sp.), head mold (Rhizopus sp.), aspergillus niger
(Aspergillus niger), Burkholderia yielding lipase (Burkholderia sp.)；Pass through genetic engineering or albumen
Lipase obtained by matter is engineered；Or commercially available lipase.
4. a kind of carrier, which is characterized in that the functional group of the carrier is and C8-15Alkane end connection primary amine groups, function
Energy group content is 1~5mmol/g, and pore-size distribution is 15~60nm, and particle diameter distribution is 0.15~0.3mm, specific surface area 150
~500m2/g；The carrier is deacidite carrier.
5. carrier as claimed in claim 4, wherein the skeleton of the carrier is polyacrylate or styrene type skeleton.
6. a kind of method for preparing immobilized lipase according to any one of claims 1 to 3, which comprises
I) carrier is provided；
Ii) lipase is provided, and zymoprotein crosslinking agent is optionally added wherein；
Contact the carrier with the lipase, so that the lipase is fixed on the carrier；
Iv) optionally drying step iii) obtained in be fixed with the carrier of lipase, to obtain the immobilized lipase.
7. method as claimed in claim 6, which is characterized in that further include carrying out following pre- place to the carrier in step i)
Reason: it with the lye and acid solution alternate immersion of 0.05mol/L~1mol/L or flushing described carrier 1~5 time, then only uses
The lye of 0.05mol/L~1mol/L impregnates or flush vehicle 1 time again, is washed with water to water and is in neutrality.
8. method as claimed in claim 6, which is characterized in that step iii) described in the ratio of carrier and the lipase be
1:3~3:1.
9. a kind of method for carrying out transesterification, esterification, acidolysis or alcoholysis reaction, the method includes using in claims 1 to 33
Described in any item immobilized lipases are as catalyst.
10. a kind of kit or product comprising immobilized lipase according to any one of claims 1 to 3.
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| CN201310656064.2A CN104694526B (en) | 2013-12-06 | 2013-12-06 | Catalytic esterification and the Sn-1,3 selectivity immobilized lipase of transesterification and preparation method thereof |
| PCT/CN2014/093082 WO2015081879A1 (en) | 2013-12-06 | 2014-12-05 | Sn-1,3 selective immobilized lipase for catalytic esterification and ester interchange and method for preparing same |
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| CN201310656064.2A CN104694526B (en) | 2013-12-06 | 2013-12-06 | Catalytic esterification and the Sn-1,3 selectivity immobilized lipase of transesterification and preparation method thereof |
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| CN106867989B (en) * | 2015-12-10 | 2022-01-21 | 丰益(上海)生物技术研发中心有限公司 | Immobilized enzyme and preparation method thereof |
| CN105368886A (en) * | 2015-12-11 | 2016-03-02 | 江苏万年长药业有限公司 | Process of using resin-immobilized halohydrin dehalogenase to catalytically synthesize (R)-4-cyan-3-hydroxy ethyl butyrate |
| JP7213184B2 (en) * | 2016-12-19 | 2023-01-26 | トランス バイオ － ディーゼル リミテッド | Enzymatic enrichment of n-3 fatty acids in the form of glycerides |
| CN107828756B (en) * | 2017-10-12 | 2020-11-27 | 广东惠尔泰生物科技有限公司 | Preparation method of Sn-1,3 specific immobilized lipase |
| CN108486094A (en) * | 2018-04-20 | 2018-09-04 | 中国科学院南海海洋研究所 | A kind of immobilized lipase and preparation method thereof |
| CN109266700A (en) * | 2018-09-27 | 2019-01-25 | 中南林业科技大学 | A method of omega-fatty acid lysophosphatide is rich in using step enzyme method preparation |
| CN110184311B (en) * | 2019-05-25 | 2022-12-16 | 华南理工大学 | Method for producing special oil OPO by using microbial fermentation |
| CN110241107A (en) * | 2019-06-11 | 2019-09-17 | 中国科学院南海海洋研究所 | A kind of method using amino resins immobilized lipase and immobilized lipase obtained by this method |
| EP4298211A1 (en) * | 2021-02-26 | 2024-01-03 | Novozymes A/S | Polystyrene/divinylbenzene particles for lipase immobilization |
| CN117230054B (en) * | 2023-11-16 | 2024-03-19 | 广东惠尔泰生物科技有限公司 | Preparation method of immobilized lipase and method for preparing UPU type glyceride by using immobilized lipase |
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| WO2011053252A1 (en) * | 2009-10-28 | 2011-05-05 | Agency For Science, Technology And Research | Polymer coated magnetic particles |
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| CN102260662B (en) * | 2010-05-24 | 2017-04-05 | 中国科学院过程工程研究所 | Carrier for immobilized enzyme and application thereof and the carrier for being fixed with enzyme |
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| CN100455668C (en) * | 2000-06-13 | 2009-01-28 | 赫尔克里士公司 | Enzyme-catalyzed polyamides and compositions and processes of preparing and using same |
| CN102105496A (en) * | 2007-09-13 | 2011-06-22 | 巴斯夫涂料有限公司 | Method of making carbamate functional materials |
| WO2011053252A1 (en) * | 2009-10-28 | 2011-05-05 | Agency For Science, Technology And Research | Polymer coated magnetic particles |
| CN102839166A (en) * | 2011-06-23 | 2012-12-26 | 丰益(上海)生物技术研发中心有限公司 | TL immobilization enzyme and application thereof |
| CN103194520A (en) * | 2013-04-22 | 2013-07-10 | 宁夏大学 | Method for preparing 4-hydroxyl tetrahydropyran derivative with optical activity through enzymatic transesterification |
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